title: Expression, purification, and partial in vitro characterization of biologically active human coagulation factor VIII light chain (A3-C1-C2) in Pichia pastoris.
abstract: Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1-A2) and light chains (A3-C1-C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.
authors.affiliation: Center for Bio Separation Technology (CBST), VIT University, Vellore, 632014, Tamil Nadu, India.
authors.forename: Sudheer Reddy
authors.initials: SR
authors.lastname: A R
authors.forename: Padikara Kutty
authors.initials: PK
authors.lastname: Satheeshkumar
authors.forename: Mookambeswaran A
authors.initials: MA
authors.lastname: Vijayalakshmi
mesh_terms: D001665:Binding Sites; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering / Q000191:economics / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010743:Phospholipids / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D010743:Phospholipids; D011994:Recombinant Proteins; D005169:Factor VIII